Composition for increasing expression of blood coagulation factor gene, comprising core-shell structured microparticles as active ingredient

ABSTRACT

The present disclosure relates to a composition for increasing the expression of a blood coagulation factor gene, which contains core-shell structured microparticles as an active ingredient. When administered in vivo along with blood coagulation factor VIII gene or a variant gene thereof, the composition for increasing the expression of a blood coagulation factor gene of the present disclosure can increase the expression of the gene by at least 30%. When administered along with a gene therapeutic agent, the composition can achieve a therapeutic effect even with a very small amount of a gene, and thus is useful.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S. patentapplication Ser. No. 16/969,040, filed Dec. 28, 2020, which is a § 371national stage entry of International Application No. PCT/KR2019/001709,filed on Feb. 12, 2019, which claims priority to Korean PatentApplication No. 10-2018-0017075, filed on Feb. 12, 2018, and KoreanPatent Application No. 10-2019-0016076, filed on Feb. 12, 2019, theentire contents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format, and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 15, 2020, isnamed G1035-17101_RevisedSequenceList.txt and is 52,733 bytes in size.

TECHNICAL FIELD

The present disclosure relates to a composition for increasing theexpression of a blood coagulation factor gene, which contains core-shellstructured microparticles as an active ingredient.

BACKGROUND ART

Hemophilia is one of severe genetic disorders. Hemophilia A is an Xgene-linked disease occurring in 1 in 5000 males. It arises from themutation of human blood coagulation factor VIII (also known as factorVIII, factor 8, FVIII or F8), which is an important plasma glycoproteinin blood clotting.

Before the 1980s, patients with hemophilia received administration ofblood coagulation factor VIII extracted from the plasma of other peoplefor treatment. But, this method had severe problems such as viralinfection, etc. In order to overcome this advantage, full-length factorVIII produced in CHO cells, etc. based on recombinant protein researchesare used for treatment since the 1980s.

The inventors of the present disclosure have researched to develop agene therapeutic agent capable of achieving therapeutic effect even witha small amount of a gene. In doing so, they have identified that, whencore-shell structured microparticles consisting of a halogenatedhydrocarbon and/or halogenated sulfur as a core and a lipid component asan outer shell are administered in vivo along with a blood coagulationfactor VIII gene, the expression of the blood coagulation factor gene isincreased remarkably, and have completed the present disclosure.

REFERENCES OF RELATED ART Patent Documents

(Patent document 001) KR 10-1542752 B.

(Patent document 002) KR 10-2018-0118659 A.

DISCLOSURE Technical Problem

The present disclosure is directed to providing a composition forincreasing the expression of a blood coagulation factor gene, whichcontains core-shell structured microparticles as an active ingredient.

The present disclosure is also directed to providing a pharmaceuticalcomposition for preventing or treating a bleeding disorder or bleeding,which contains the composition described above.

Technical Solution

The present disclosure provides a composition for increasing theexpression of a blood coagulation factor gene, which contains core-shellstructured microparticles as an active ingredient, wherein the core is ahalogenated hydrocarbon, halogenated sulfur or a mixture thereof as abiocompatible gas, and the shell is a lipid or a derivative thereof, andthe blood coagulation factor gene is one or more gene selected from ahuman blood coagulation factor VIII gene or a variant gene thereof.

In an exemplary embodiment of the present disclosure, the biocompatiblegas may be selected from sulfur hexafluoride, octafluoropropane,bromochlorodifluoromethane, chlorodifluoromethane,dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane,chloropentafluoroethane, dichlorotetrafluoroethane and a mixturethereof.

In an exemplary embodiment of the present disclosure, the halogenatedhydrocarbon may be a perfluorinated hydrocarbon.

In an exemplary embodiment of the present disclosure, the perfluorinatedhydrocarbon may be perfluoromethane, perfluoroethane, perfluoropropane,perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane,perfluoropropene, perfluorobutene, perfluorobutadiene,perfluorobut-2-ene, perfluorocyclobutane, perfluoromethylcyclobutane,perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclopentane, perfluoromethylcyclohexane,perfluoromethylcyclohexane, perfluoromethylcyclohexane or a mixturethereof.

In an exemplary embodiment of the present disclosure, the lipid may beone or more selected from a group consisting of a simple lipid, aphospholipid, a glyceroglycolipid, a sphingoglycolipid, a cholesteroland a cationic lipid.

In an exemplary embodiment of the present disclosure, the phospholipidmay be selected from a group consisting of a phosphatidylcholinederivative, a phosphatidylethanolamine derivative, a phosphatidylserinederivative, a diacetylated phospholipid, L-α-dioleylphosphatidylethanolamine, diolein, phosphatidic acid,phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine,sphingomyelin, a polyethylene glycolated phospholipid, egg yolklecithin, soy lecithin and a hydrogenated phospholipid.

In an exemplary embodiment of the present disclosure, theglyceroglycolipid may be selected from a group consisting ofsulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyldiglyceride, galactosyl diglyceride and glycosyl diglyceride.

In an exemplary embodiment of the present disclosure, thesphingoglycolipid may be galactosyl cerebroside, lactosyl cerebroside organglioside.

In an exemplary embodiment of the present disclosure, the cationic lipidmay be selected from a group consisting of1,2-dioleoyl-3-trimethylammonium propane (DOTAP),N-(2,3-dioleyloxypropan-1-yl)-N,N,N-trimethylammonium chloride (DOTMA),2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE),1,2-dioleoyloxypropyl-3-diethylhydroxyethylammonium bromide (DORIE) and3β-[N—(N′N′-dimethylaminoethylhy)carbamoyl]cholesterol (DC-Chol).

In an exemplary embodiment of the present disclosure, the bloodcoagulation factor VIII may be composed of an amino acid sequencerepresented by SEQ ID NO 1, and the variant of blood coagulation factorVIII may be composed of an amino acid sequence represented by SEQ ID NO3.

In an exemplary embodiment of the present disclosure, the compositionmay increase the expression of the blood coagulation factor gene by 30%or more.

The present disclosure also provides a pharmaceutical composition forpreventing or treating a bleeding disorder or bleeding, which containsthe composition described above and a human blood coagulation factorVIII gene or a variant gene thereof.

In an exemplary embodiment of the present disclosure, the bleedingdisorder may be hemophilia A, hemophilia induced or complicated by aninhibitory antibody against blood coagulation factor VIII or bloodcoagulation factor VIIIa, or hemophilia B.

In an exemplary embodiment of the present disclosure, the bleedingdisorder or bleeding may be one selected from a group consisting ofneonatal coagulopathy, severe liver disease, thrombocytopenia,congenital deficiency of factor V, VII, X or XI, and von Willebranddisease having an inhibitor against von Willebrand factor.

In an exemplary embodiment of the present disclosure, the bleeding maybe caused by blood loss associated with high-risk surgical procedures,traumatic blood loss, blood loss caused by bone marrow transplantationor blood loss caused by cerebral hemorrhage.

Advantageous Effects

A composition for increasing the expression of a blood coagulationfactor gene of the present disclosure may increase the expression of theblood coagulation factor gene by at least 30% when administered in vivoalong with blood coagulation factor VIII gene or a variant gene thereof(e.g., a polynucleotide encoding the gene or a vector including thesame). Accordingly, it is useful since a therapeutic effect can beachieved with a very small amount of the gene when the composition isadministered along with a gene therapeutic agent.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically illustrates a process of designing a bloodcoagulation factor VIII variant (F8M) according to an exemplaryembodiment of the present disclosure.

FIG. 2 shows the cleavage map of a pGP vector according to an exemplaryembodiment of the present disclosure.

FIG. 3 compares the expression level of the F8 protein in mousedepending on the administration of pGP-F8M (gene only), a pharmaceuticalcomposition of a control group (F8M-JetPEI) and pharmaceuticalcompositions according to Example (F8M-MP1 and F8M-MP2).

FIG. 4 compares the expression level of the F9 protein in mousedepending on the administration of pGP-F9 (gene only) and pharmaceuticalcompositions according to Comparative Example (F9-MP1 and F9-MP2).

FIG. 5 schematically shows the domain structure constituting human bloodcoagulation factor VIII according to an exemplary embodiment of thepresent disclosure.

BEST MODE

Hereinafter, the present disclosure is described in detail.

In an aspect, the present disclosure provides a composition forincreasing the expression of a blood coagulation factor gene, whichcontains core-shell structured microparticles as an active ingredient,wherein the core is a halogenated hydrocarbon, halogenated sulfur or amixture thereof as a biocompatible gas, and the shell is a lipid or aderivative thereof, and the blood coagulation factor gene is one or moregene selected from a human blood coagulation factor VIII gene or avariant gene thereof.

Core

In the present disclosure, the “core” may be composed of a halogenatedhydrocarbon, halogenated sulfur or a mixture thereof as a biocompatiblegas.

The biocompatible gas may be sulfur hexafluoride, octafluoropropane,bromochlorodifluoromethane, chlorodifluoromethane,dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane,chloropentafluoroethane, dichlorotetrafluoroethane or a mixture thereof.

Specifically, the halogenated hydrocarbon may be a perfluorinatedhydrocarbon.

The perfluorinated hydrocarbon may be perfluoromethane, perfluoroethane,perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane,perfluoroheptane, perfluoropropene, perfluorobutene, perfluorobutadiene,perfluorobut-2-ene, perfluorocyclobutane, perfluoromethylcyclobutane,perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclopentane, perfluoromethylcyclohexane,perfluoromethylcyclohexane, perfluoromethylcyclohexane or a mixturethereof.

Specifically, the biocompatible gas of the present disclosure may besulfur hexafluoride or perfluorobutane.

Shell

In the present disclosure, the “shell” may be composed of a lipid or aderivative thereof.

The lipid may be one or more selected from a group consisting of asimple lipid, a phospholipid, a glyceroglycolipid, a sphingoglycolipid,a cholesterol and a cationic lipid. Specifically, it may be aphospholipid.

The phospholipid may be a phosphatidylcholine derivative, aphosphatidylethanolamine derivative, a phosphatidylserine derivative,diacetylated phospholipid, L-α-dioleyl phosphatidylethanolamine,diolein, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol,lysophosphatidylcholine, sphingomyelin, polyethylene glycolatedphospholipid, egg yolk lecithin, soy lecithin, a hydrogenatedphospholipid, etc.

The glyceroglycolipid may be sulfoxyribosyl glyceride, diglycosyldiglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyldiglyceride, etc.

The sphingoglycolipid may be galactosyl cerebroside, lactosylcerebroside, ganglioside, etc.

And, the cationic lipid may be 1,2-dioleoyl-3-trimethylammonium propane(DOTAP), N-(2,3-dioleyloxypropan-1-yl)-N,N,N-trimethylammonium chloride(DOTMA),2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE),1,2-dioleoyloxypropyl-3-diethylhydroxyethylammonium bromide (DORIE),3β-[N—(N′N′-dimethylaminoethylhy)carbamoyl]cholesterol (DC-Chol), etc.

Microparticles

The microparticles of the present disclosure are stabilized by the shellwhich surrounds the core gas. The shell retards the diffusion of a gasto nearby liquid and prevents fusion between the microparticles.

When administered in vivo, the microparticle retains its shape until itreaches a target cell or tissue, and releases the gas as it is destroyednear the target cell or tissue. The released gas may cause change in thecell membrane of the target cell and may facilitate the entry of thegrowth factor gene into the cytoplasmic environment of the target cellvia the jet force of the gas.

The microparticles may have an average diameter of 1-10 μm, specifically2-8 μm, more specifically 2-4 μm.

Composition for Increasing Gene Expression

Recently, there have been many researches on core-shell microparticleshaving a gas as a core. In the previous researches, the effect ofincreasing gene expression was not achieved unless ultrasound wasirradiated together with the microparticles.

Specifically, Sang-Chol Lee et al., Korean Circulation J 2006; 36:32-38; “Enhancement of Gene Delivery into Mouse Skeletal Muscle withMicrobubble Destruction by Low-Frequency Ultrasound” discloses that theeffect of increasing gene expression is not achieved when only aluciferase gene-microparticles mixture is injected without irradiationof ultrasound.

Z P Shen et al., Gene Therapy (2008) 15, 1147-1155; “Ultrasound withmicrobubbles enhances gene expression of plasmid DNA in the liver viaintraportal delivery” also discloses that the effect of increasing geneexpression is not achieved when only a luciferase gene-microparticlesmixture is injected without irradiation of ultrasound.

In addition, Xingsheng Li et al., J Ultrasound Med 2008; 27: 453-460;“Experimental Research on Therapeutic Angiogenesis Induced by HepatocyteGrowth Factor Directed by Ultrasound-Targeted Microbubble Destruction inRats” discloses that the effect of increasing gene expression is notachieved when only a HGF gene-liposome microparticles mixture isinjected without irradiation of ultrasound.

However, the inventors of the present disclosure have identified that,while studying on the use of microparticles, the expression level of ablood coagulation factor gene, particularly a blood coagulation factorVIII gene, is increased remarkably even without ultrasound irradiationwhen the gene is injected along with the microparticles according to thepresent disclosure, and have completed the present disclosure.Meanwhile, as described specifically in the test example describedlater, the effect of increasing gene expression was not achieved withthe microparticles for blood coagulation factor genes other than theblood coagulation factor VIII gene or a variant gene thereof (e.g.,blood coagulation factor IX (also known as factor IX, factor 9, FIX orF9)).

When administered in vivo along with a blood coagulation factor VIIIgene or a variant gene thereof, the composition for increasing geneexpression of the present disclosure can increase the expression levelof the blood coagulation factor gene by at least 30%.

In the examples described below, it was verified that, when administeredalong with a human blood coagulation factor VIII variant gene intomouse, the composition for increasing gene expression of the presentdisclosure increases the expression level of the blood coagulationfactor VIII gene remarkably. More specifically, it was verified that,when the composition for increasing gene expression of the presentdisclosure and the blood coagulation factor VIII variant gene areadministered together into an animal model, the expression level of theblood coagulation factor VIII gene is increased by 40% or more. Incontrast, for a blood coagulation factor IX gene, the expression of thegene was hardly increased even when the composition for increasing geneexpression of the present disclosure was administered together intomouse.

That is to say, it seems that the composition for increasing geneexpression of the present disclosure is effective in increasing theexpression level of the human blood coagulation factor gene specificallyonly when administered along with one or more gene selected from humanblood coagulation factor VIII gene and a variant gene thereof, fromamong blood coagulation factors.

The composition may further contain a pharmaceutical adjuvant such as astabilizer, a buffer, a salt for control of osmotic pressure, anexcipient, an antiseptic, etc. or other therapeutically usefulsubstances, and may be prepared into various formulations for oral orparenteral administration, specifically for parenteral administration,according to common methods. Specifically, a formulation for parenteraladministration may be typically an injection formulation in the form ofan isotonic aqueous solution or suspension. Alternatively, thecomposition may be prepared into a powder and then suspended in asolvent immediately before administration.

In the composition for increasing gene expression of the presentdisclosure, the content of the microparticles may be 0.5-2,000 μL/mL,specifically 1-1,000 μL/mL or 5-2,000 μg/mL, specifically 10-1,000μg/mL, although not being particularly limited.

If the content of the microparticles is outside the above range, thedesired effect cannot be achieved.

Specifically, the composition may be administered as a mixture with agene to achieve a better effect.

Gene

The composition for increasing gene expression according to the presentdisclosure may further increase the expression and efficiency of geneswhen administered along with the following genes.

Blood Coagulation Factor VIII

In the present disclosure, the terms “blood coagulation factor VIII”,“factor VIII”, “FVIII” and “F8” are used interchangeably. Mature humanblood coagulation factor VIII is composed of 2351 amino acids (includingsignal peptides), which are arranged in a domain structure shown in FIG.5 .

In FIG. 5 , a1, a2 and a3 are acidic domains. The acidic domain a3 isknown to be involved in the binding of the blood coagulation factor VIIImolecule to a von Willebrand factor (vWF), which plays an important rolein blood coagulation. In the process of secretion, the blood coagulationfactor VIII is cleaved between the B-domain and the a3 acidic domain,resulting in a heterodimeric polypeptide. The blood coagulation factorVIII heterodimer consists of a light chain (including A3, C1 and C2) anda heavy chain of variable size (including A1, A2 and B). The heavy chainis heterogeneous due to limited proteolysis in the B-domain. In case ofheterodimeric B-domain-deleted blood coagulation factor VIII, the “heavychain” includes A1 and A2 but the B-domain is deleted partially orwholly.

The amino acid sequence of mature wild-type human blood coagulationfactor VIII is shown in SEQ ID NO 1. The reference of an amino acidposition of a specific sequence means the position of the amino acid inthe VIII wild-type protein and does not exclude the presence ofmutations (e.g., deletion, insertion and/or substitution) at otherpositions in the sequence. A DNA sequence encoding SEQ ID NO 1 isrepresented by SEQ ID NO 2.

“Blood coagulation factor VIII” includes not only wild-type bloodcoagulation factor VIII but also derivatives of the wild-type bloodcoagulation factor VIII having the procoagulant activity of thewild-type blood coagulation factor VIII. The derivative may have asequence with deletion, insertion and/or addition as compared to theamino acid sequence of the wild-type blood coagulation factor VIII.Specifically, the derivative may be a molecule of blood coagulationfactor VIII with all or part of the B-domain deleted. Throughout thepresent disclosure, the positions amino acids always refer to thepositions of individual amino acids in the full-length mature wild-typeblood coagulation factor VIII (including signal peptides).

Blood Coagulation Factor VIII Variant

In the present disclosure, the term “variant” includes conservative ornon-conservative substitution, insertion or deletion of an amino acidsequence, a nucleic acid sequence, etc., and such change does notsubstantially alter the active site or active domain that confers thebiological activity of FVIII.

The blood coagulation factor VIII variant according to the presentdisclosure is a single-chain blood coagulation factor VIII variant inwhich amino acids Asp784 to Arg1671 are deleted from blood coagulationfactor VIII represented by SEQ ID NO 1. The blood coagulation factorVIII variant is one in which a part of the B-domain (residues 784-1667)and a part of the a3 domain (residues 1668-1671) are deleted, and thevariant exhibits enhanced protein expression and improved bloodcoagulation activity and stability as compared to blood coagulationfactor VIII and B-domain-deleted blood coagulation factor VIII. Theblood coagulation factor VIII variant has an amino acid sequencerepresented by SEQ ID NO 3.

The “single-chain blood coagulation factor VIII” means a bloodcoagulation factor VIII molecule which exists as a single polypeptidechain without being cleaved into tow chains (e.g., a heavy chain and alight chain) by proteolysis during secretion from a cell expressing theblood coagulation factor VIII molecule.

In addition, the blood coagulation factor VIII variant may be expressedas a polynucleotide encoding the amino acid sequence.

Polynucleotide

In the present disclosure, the term “polynucleotide(s)” refers to anypolyribonucleotide or polydeoxyribonucleotide which may be an unmodifiedRNA or DNA or a modified RNA or DNA. The polynucleotide of the presentinvention may be a single-chain DNA or RNA. As used herein, the term“polynucleotide(s)” includes a DNA or RNA containing modified basesand/or unique bases such as inosine. It is obvious that variousmodifications can be made to the DNA or RNA to serve a known usefulpurpose. As used herein, the term “polynucleotide(s)” includes suchchemically, enzymatically or metabolically modified polynucleotide(s).

Those skilled in the art will appreciate that the blood coagulationfactor VIII variant may be encoded by several polynucleotides due to thedegeneracy of the genetic code. In other words, a polynucleotidesequence encoding the blood coagulation factor VIII variant that can beused in the present invention is interpreted to include a nucleotidesequence showing substantial identity to the amino acid sequence.

Plasmid

When the composition for increasing gene expression according to thepresent disclosure is administered along with a plasmid containing asingle-chain polynucleotide encoding the gene, the expression efficiencyand efficacy of the gene may be increased further.

In the present disclosure, the term “plasmid” generally refers to acircular DNA molecule formed by being operably linked to a vector sothat an exogenous gene can be expressed in a host cell. However, theplasmid can be used as a vector that is degraded by specific restrictionenzymes by gene recombination to incorporate a desired gene. Thus, theterms plasmid and vector are used interchangeably herein, and thoseskilled in the art of genetic engineering will fully understand theirmeanings even if they are not distinguished by names.

In the present disclosure, the term “vector” refers to a DNA molecule asa carrier that can stably transport an exogenous gene into a host cell.To be a useful vector, it must be replicable, have a way to enter thehost cell, and be equipped with a means to detect its presence.

Expression

Expression Vector

The composition for increasing gene expression according to the presentdisclosure can further increase the expression and efficiency of a genewhen administered along with expression vectors including single-chainpolynucleotides encoding the gene.

In the present disclosure, the term “expression” refers to generation ofthe gene in a cell.

In the present disclosure, the term “expression vector” refers to avector capable of expressing a target gene in a suitable host, and meansa gene construct including an essential regulatory element operablylinked to express a gene insert.

In the present disclosure, the term “operably linked” means that anucleic acid expression-regulating sequence and a polynucleotideencoding a target gene are functionally linked to perform a generalfunction. For example, a promoter and a polynucleotide encoding the genemay be operably linked to affect the expression of the polynucleotide.The operable linkage to a recombinant vector may be prepared using agenetic recombinant technique well known in the art, and site-specificDNA cleavage and ligation may be achieved using enzymes generally knownin the art.

The expression vector of the present disclosure may be prepared using aplasmid, a vector or a viral vector, although not being limited thereto.An appropriate expression vector may include an expression-regulatingelement such as a promoter, an operator, a start codon, a stop codon, apolyadenylation signal, an enhancer, etc. and may be prepared variouslyaccording to purposes. The promoter of the vector may be constitutive orinducible. Since a plasmid is the most commonly used form of a vector atpresent, the terms “plasmid” and “vector” are used sometimesinterchangeably in the present disclosure. For the purpose of thepresent disclosure, it is preferred to use a plasmid vector. A typicalplasmid vector that can be used for this purpose has a structureincluding (a) a replication origin for effective replication intoseveral to hundreds of plasmid vectors per host cell and (b) arestriction enzyme site into which a fragment of foreign DNA can beinserted. Even if a proper restriction enzyme site does not exist, thevector and foreign DNA can be easily ligated using syntheticoligonucleotide adaptors or linkers according to common methods.

A vector used for overexpression of a gene according to the presentdisclosure may be an expression vector known in the art. A frameworkvector that may be used in the present disclosure may be selected from agroup consisting of pCDNA3.1, pGP, pEF, pVAX, pUDK, pCK, pQE40, pT7,pET/Rb, pET28a, pET-22b(+) and pGEX, although not being particularlylimited thereto. Specifically, use of a vector selected from a groupconsisting of pGP, pEF, pCK, pUDK and pVAX may be preferred in terms ofeffect.

In a specific exemplary embodiment, the expression vector of the presentdisclosure may be an expression vector including a pGP vector having acleavage map of FIG. 2 .

Pharmaceutical Composition

In another aspect, the present disclosure provides a pharmaceuticalcomposition for preventing or treating a bleeding disorder or bleeding,which contains the composition for increasing gene expression of thepresent disclosure and the blood coagulation factor VIII gene or avariant gene thereof.

The bleeding disorder may be hemophilia A, hemophilia induced orcomplicated by an inhibitory antibody against blood coagulation factorVIII or blood coagulation factor VIIIa, or hemophilia B. In addition,the bleeding disorder or bleeding may be one selected from a groupconsisting of neonatal coagulopathy, severe liver disease,thrombocytopenia, congenital deficiency of factor V, VII, X or XI, andvon Willebrand disease having an inhibitor against von Willebrandfactor.

And, the bleeding may be one caused by blood loss associated withhigh-risk surgical procedures, traumatic blood loss, blood loss causedby bone marrow transplantation or blood loss caused by cerebralhemorrhage.

The pharmaceutical composition for preventing or treating a bleedingdisorder or bleeding may be usefully used to prevent or treat a bleedingdisorder or bleeding since it exhibits superior therapeutic effectbecause gene expression is increased in vivo even with a small amount ofthe blood coagulation factor VIII gene or a variant gene thereof.

In the pharmaceutical composition of the present disclosure, thecomposition for increasing gene expression and the blood coagulationfactor VIII gene or a variant gene thereof may be contained with avolume ratio of 1:0.5-30 (w/v).

The pharmaceutical composition of the present disclosure may be for genetherapy.

Formulation

The pharmaceutical composition of the present disclosure may be preparedinto pharmaceutical formulations for therapeutic purposes.

Pharmaceutical carriers and excipients and suitable pharmaceuticalformulations are known in the art (e.g., “Pharmaceutical FormulationDevelopment of Peptides and Proteins”, Frokjaer et al., Taylor & Francis(2000) or “Handbook of Pharmaceutical Excipients”, 3rd edition, Kibbe etal., Pharmaceutical Press (2000)). In particular, the pharmaceuticalcomposition of the present disclosure may be formulated as a lyophilizedform or a stable liquid form. The composition of the present disclosuremay be freeze-dried through various procedures known in the art. Thefreeze-dried formulation is reconstituted by adding one or morepharmaceutically acceptable diluent such as sterile water for injectionor sterile physiological saline.

The formulation of the composition is delivered to a subject via anypharmaceutical suitable means of administration. Various known deliverysystems may be used to deliver the composition through any convenientroutes. Mainly, the composition of the present disclosure isadministered systemically. For systemic administration, the compositionof the present disclosure is formulated for parenteral (e.g.,intravenous, subcutaneous, intramuscular, intraperitoneal,intracerebral, intrapulmonary, intranasal or transdermal) delivery orenteral (e.g., oral, vaginal or rectal) delivery according to commonmethods. The most preferred routes of administration are intravenous andintramuscular routes. These formulations can be administeredcontinuously by infusion or by bolus injection. Some formulationsencompass slow release systems.

The composition of the present disclosure is administered to a patientin a therapeutically effective dose, meaning a dose that is sufficientto produce the desired effect, preventing or lessening the severity orspread of the condition or indication being treated without reaching adose which produces intolerable adverse effects. The exact dose dependson many factors such as the indication, formulation, mode ofadministration, etc. and has to be determined through preclinical andclinical trials for each respective indication.

The pharmaceutical composition of the present disclosure may beadministered either alone or in combination with other therapeuticagents. These agents may be incorporated as a part of the samepharmaceutical.

Treatment Method

The present disclosure also relates to a method for treating a subjectsuffering from a bleeding disorder such as hemophilia A, hemophilia B oracquired hemophilia or a subject suffering from a chronic disease orliver disease. The treatment method may include a step of administeringan effective amount of the pharmaceutical composition of the presentdisclosure to the subject.

According to an exemplary embodiment of the present disclosure, the geneof the present disclosure such as blood coagulation factor VIII gene ora variant gene thereof may be administered at a dose of 10 ng to 100 mg.When the administration of the blood coagulation factor VIII, a variantthereof or a polynucleotide encoding the same is repeated more thanonce, the administration dose may be the same or different for eachadministration.

Hereinafter, the present disclosure is described in more detail throughspecific examples. However, the examples are only for illustrating thepresent disclosure in more detail and it will be obvious to those ofordinary skill in the art that the scope of the present disclosure isnot limited by them.

Examples

Preparation of Materials

Genes

Blood Coagulation Factor VIII (F8)

A gene full-length blood coagulation factor VIII (F8) represented by SEQID 1 was synthesized by Genscript (USA).

Variant of Blood Coagulation Factor VIII (F8M)

A full-length blood coagulation factor VIII gene represented by SEQ IDNO 1 was used as a template, and fragment 1 represented by SEQ ID NO 4was prepared through polymerase chain reaction (PCR) using primers 1 and2. The PCR was performed by mixing 2 μL of the template DNA, 1 μL of 10pmol/μL primer each, 2.5 μL of 2.5 mM dNTP, 1 μL of a pfu enzyme mix(Enzynomics, Korea), 2.5 μL of a 10×buffer, and 50 μL of sterilizedtriply distilled water. The solution was reacted for 30 seconds at 95°C., 30 seconds at 60° C. and 30 seconds at 72° C. for 40 cycles. Usingprimers 3 and 4, fragment 2 represented by SEQ ID NO 5 was prepared byPCR as in the same manner as described above. Subsequently, thefragments 1 and 2 were subjected to overlapping PCR using primers 5 and6, thereby preparing a single-chain blood coagulation factor VIIIvariant. In the overlapping PCR, 2 μL of each DNA fragment was added,and reaction time and method were the same as described above in thePCR. FIG. 1 schematically illustrates the process of designing the bloodcoagulation factor VIII variant (F8M) according to an exemplaryembodiment of the present disclosure.

The primers are summarized in Table 1 below.

TABLE 1 Primer No. Primer name Base sequence 1 F8(F)ATGCAAATAGAGCTCTCCACCTGCTTCTTT 2 F8M-1(R)TCTGACTGAAGAGTAGTATTTTCTGGAATTGTGGT GGCATTAAAT 3 F8M-2(F)CCACAATTCCAGAAAATACTACTCTTCAGTCAGAT CAAGAGGAAATTGAC 4 F8(R)TCAGTAGAGGTCCTGTGCCTCGCAGCCCAG 5 F8 final(F)GCTAGCATGCAAATAGAGCTCTCCACCTGC 6 F8 final(R)GCGGCCGCTCAGTAGAGGTCCTGTGCCTCGCA

Blood Coagulation Factor IX (F9)

A gene of full-length blood coagulation factor IX (see GenBank basesequence FR846239.1) represented by SEQ ID NO 6 was synthesized byBionics (Korea).

After synthesizing pCK plasm ids referring to the literature Lee et al.(Lee Y, et al. Improved expression of vascular endothelial growth factorby naked DNA in mouse skeletal muscles: implication for gene therapy ofischemic diseases. Biochem. Biophys. Res. Commun. 2000; 272(1):230-235), PCR was conducted in the same manner described in theliterature using primers 7 and 8 of Table 2 below. After reacting theobtained fragments with EcoRI enzyme at 37° C. for 1 hour, DNA waspurified using an Expin Gel SV (GeneAll, Korea) kit. Then, afterconducting ligation for 30 minutes using T4 ligase, the DNA wasincubated overnight with E. coli. Next day, after isolating DNA from thecolony through mini-prep, pGP plasmids represented by SEQ ID NO 7 wereobtained. FIG. 2 shows the cleavage map of the pGP vector according toan exemplary embodiment of the present disclosure.

TABLE 2 Primer No. Primer name Base sequence 7 pGP(F)GACGAATTCACGCGTCTCGAGGCGGCCGCTCTA GAGGGCCCGTTTAAA 8 pGP(R)GACGAATTCGTCGACGGATCCGCTAGCAAGCTT CGTGTCAAGGACGGT

Preparation Example

Preparation of Plasmid DNAs Including Genes

Each of the genes and each of the pGP plasmids prepared above werecleaved with NheI and NotI enzymes for 1 hour and fragments wereseparated by conducting electrophoresis on agarose gel. The separatedfragments were ligated for 30 minutes using T4 ligase and then incubatedovernight with E. coli. Next day, DNA was isolated from the colonythrough mini-prep, and then digested with NheI and NotI. The cloned DNAwas incubated overnight with an E. coli supernatant digested withrestriction enzymes in a 4-L flask in the presence of kanamycin. PlasmidDNAs produced using an Endofree Giga prep. kit (Qiagen, USA) were usedin animal experiments. The prepared plasmid DNAs are summarized in Table3.

TABLE 3 Gene Plasmid DNA Blood coagulation factor VIII (F8) pGP-F8 Bloodcoagulation factor VIII variant (F8M) pGP-F8M Blood coagulation factorIX (F9) pGP-F9

Examples Example: Preparation of Pharmaceutical Composition ContainingComposition for Increasing Expression of Gene and F8M (F8M-MP1 andF8M-MP2)

Composition for Increasing Gene Expression

Core-shell structured microparticles with a reference code of 62400210,having an average diameter of about 2.5 μm and composed of sulfurhexafluoride as a core and a lipid as a shell, were purchased fromBracco Imaging Korea (MP1). In addition, core-shell structuredmicroparticles with a reference code of 646300210, having an averagediameter of about 2.4-3.6 μm and composed of perfluorobutane as a coreand a shell including a lipid and a surfactant, were purchased from GEHealthcare Korea (MP2).

Preparation of Pharmaceutical Composition Containing Composition forIncreasing Gene Expression and F8M

Pharmaceutical compositions F8M-MP1 and F8M-MP2 were prepared by mixing15 μL of the compositions for increasing gene expression (MP1 and MP2),respectively, with the pGP-F8M prepared above (70 μg/35 μL).

Comparative Example: Preparation of Pharmaceutical CompositionContaining Composition for Increasing Gene Expression and F9 (F9-MP1 andF9-MP2)

Composition for Increasing Gene Expression

Compositions for increasing gene expression were prepared in the samemanner as in Example.

Preparation of Pharmaceutical Composition Containing Composition forIncreasing Gene Expression and F9

Pharmaceutical compositions F9-MP1 and F9-MP2 were prepared by mixing 15μL of the compositions for increasing gene expression (MP1 and MP2),respectively, with the pGP-F9 prepared above (70 μg/35 μL).

Control Group

Composition for Increasing Gene Expression

A suspension (corresponding to a composition for increasing geneexpression) was prepared by mixing 128 μL of in vivo JetPEI (Polyplus,USA) with 2 mL of 5% glucose according to the manufacturer's manual.

Preparation of Pharmaceutical Composition Containing Composition forIncreasing Gene Expression and Each Gene

A pharmaceutical compositions F8M-JetPEI was prepared by mixing 15 μL ofthe composition with the pGP-F8M prepared above (70 μg/35 μL).

Test Example Test Example: Expression Level of Protein in Mouse

Each of the pharmaceutical compositions according to the control group,example and comparative example was injected into the lower calf muscleof C57bl/6 mouse with 75 μg/50 μL/leg.

On day 7 after the administration, the mouse was sacrificed and themuscle at the injected area was excised. Then, total proteins wereisolated after grinding the excised muscle was using liquid nitrogen anda protein extraction kit (Cell Biolabs, USA). The amounts of theisolated total proteins were measured using a DC protein assay kit(Bio-Rad laboratories, USA).

For measurement of the F8 protein, the expression level of each gene wasmeasured using an ELISA kit (Stago Asserchrom VIII:Ag, France) for thesame amount of protein. The result is shown in FIG. 3 .

From FIG. 3 , it can be seen that the administration of thepharmaceutical composition according to the present disclosure (Example)resulted in a statistically significantly higher expression level ascompared to the administration of the gene only or the administration ofthe composition of the control group. Specifically, the groupadministered with the F8M-MP1 of Example showed 44% higher expressionlevel as compared to group administered with the gene only (pGP-F8M),and the group administered with F8M-MP2 showed 116% higher expressionlevel as compared to group administered with the gene only.

For measurement of the F9 protein, the expression level of each gene wasmeasured using an ELISA kit (Abcam, USA) for the same amount of protein.The result is shown in FIG. 4 .

From FIG. 4 , it can be seen that the administration of the compositionof Comparative Example hardly resulted in increased gene expression ascompared to the administration of the gene only.

That is to say, from FIG. 3 and FIG. 4 , it can be seen that theadministration of the compositions according to the present disclosure(Example) results in a significantly higher expression level as comparedto the administration of the gene only or the administration of thecomposition of the control group or comparative example.

Although the present disclosure was illustrated with the specificexemplary embodiments described above, various modifications or changescan be made thereto without departing from the subject matter and scopeof the present disclosure. In addition, such modifications or changeswithin the subject matter of the present disclosure are included in thescope of the appended claims.

1. A method for increasing the expression of a blood coagulation factorgene in a subject in need thereof comprising administering to thesubject the blood coagulation factor gene and a composition comprisingcore-shell structured microparticles, wherein the core is sulfurhexafluoride or perfluorobutane as a biocompatible gas, and the shell isa lipid or a derivative thereof, and the blood coagulation factor geneis one or more genes selected from a human blood coagulation factor VIIIgene or a variant gene thereof.
 2. The method for increasing theexpression of a blood coagulation factor gene according to claim 1,wherein the lipid is one or more selected from a group consisting of asimple lipid, a phospholipid, a glyceroglycolipid, a sphingoglycolipid,a cholesterol and a cationic lipid.
 3. The method for increasing theexpression of a blood coagulation factor gene according to claim 2,wherein the phospholipid is selected from a group consisting of aphosphatidylcholine derivative, a phosphatidylethanolamine derivative, aphosphatidylserine derivative, a diacetylated phospholipid, L-α-dioleylphosphatidylethanolamine, diolein, phosphatidic acid,phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine,sphingomyelin, a polyethylene glycolated phospholipid, egg yolklecithin, soy lecithin and a hydrogenated phospholipid.
 4. The methodfor increasing the expression of a blood coagulation factor geneaccording to claim 2, wherein the glyceroglycolipid is selected from agroup consisting of sulfoxyribosyl glyceride, diglycosyl diglyceride,digalactosyl diglyceride, galactosyl diglyceride and glycosyldiglyceride.
 5. The method for increasing the expression of a bloodcoagulation factor gene according to claim 2, wherein thesphingoglycolipid is galactosyl cerebroside, lactosyl cerebroside organglioside.
 6. The method for increasing the expression of a bloodcoagulation factor gene according to claim 2, wherein the cationic lipidis selected from a group consisting of 1,2-dioleoyl-3-trmethylammoniumpropane (DOTAP), N-(2,3-dioleyloxypropan-1-yl)-N,N,N-trmethylammoniumchloride (DOTMA),2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE),1,2-dioleoyloxypropyl-3-diethylhydroxyethylammonium bromide (DORIE) and3β-[N—(N′N′-dimethylaminoethylhy)carbamoyl]cholesterol (DC-Chol).
 7. Themethod for increasing the expression of a blood coagulation factor geneaccording to claim 1, wherein the blood coagulation factor VIII iscomposed of an amino acid sequence represented by SEQ ID NO 1, and thevariant of blood coagulation factor VIII is composed of an amino acidsequence represented by SEQ ID NO
 3. 8. The method for increasing theexpression of a blood coagulation factor gene according to claim 1,wherein the composition increases the expression of the bloodcoagulation factor gene by 30% or more when the composition isadministered along with the human blood coagulation factor VIII gene ora variant gene thereof.
 9. A method for preventing or treating ableeding disorder or bleeding in a subject, comprising administering tothe subject the blood coagulation factor gene and a compositioncomprising core-shell structured microparticles, wherein the core issulfur hexafluoride or perfluorobutane as a biocompatible gas, and theshell is a lipid or a derivative thereof, and the blood coagulationfactor gene is one or more genes selected from a human blood coagulationfactor VIII gene or a variant gene thereof.
 10. The method forpreventing or treating a bleeding disorder or bleeding according toclaim 9, wherein the bleeding disorder is hemophilia A, hemophiliainduced or complicated by an inhibitory antibody against bloodcoagulation factor VIII or blood coagulation factor VIIIa, or hemophiliaB.
 11. The method for preventing or treating a bleeding disorder orbleeding according to claim 9, wherein the bleeding disorder or bleedingis one of neonatal coagulopathy, severe liver disease, thrombocytopenia,congenital deficiency of factor V, VII, X or XI, and von Willebranddisease having an inhibitor against von Willebrand factor.
 12. Themethod for preventing or treating a bleeding disorder or bleedingaccording to claim 9, wherein the bleeding is caused by blood lossassociated with high-risk surgical procedures, traumatic blood loss,blood loss caused by bone marrow transplantation or blood loss caused bycerebral hemorrhage.